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BACKGROUND: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind. METHODS: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40). FINDINGS: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcÎ³RI and FcÎ³RIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40. INTERPRETATION: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection. FUNDS: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Ebola virus (EBOV) disease can result in a range of symptoms anywhere from virtually asymptomatic to severe hemorrhagic fever during acute infection. Additionally, spans of asymptomatic persistence in recovering survivors is possible, during which transmission of the virus may occur. In acute infection, substantial cytokine storm and bystander lymphocyte apoptosis take place, resulting in uncontrolled, systemic inflammation in affected individuals. Recently, studies have demonstrated the presence of EBOV proteins VP40, glycoprotein (GP), and nucleoprotein (NP) packaged into extracellular vesicles (EVs) during infection. EVs containing EBOV proteins have been shown to induce apoptosis in recipient immune cells, as well as contain pro-inflammatory cytokines. In this manuscript, we review the current field of knowledge on EBOV EVs including the mechanisms of their biogenesis, their cargo and their effects in recipient cells. Furthermore, we discuss some of the effects that may be induced by EBOV EVs that have not yet been characterized and highlight the remaining questions and future directions.

RESUMO

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4+ T cell responses but limited CD8+ T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity.

RESUMO

The 2013-2015 outbreak of Ebola virus disease in Guinea, Liberia, and Sierra Leone was unprecedented in the number of documented cases, but there have been few published reports on immune responses in clinical cases and their relationships with the course of illness and severity of Ebola virus disease. Symptoms of Ebola virus disease can include severe headache, myalgia, asthenia, fever, fatigue, diarrhea, vomiting, abdominal pain, and hemorrhage. Although experimental treatments are in development, there are no current U.S. Food and Drug Administration-approved vaccines or therapies. We report a detailed study of host gene expression as measured by microarray in daily peripheral blood samples collected from a patient with severe Ebola virus disease. This individual was provided with supportive care without experimental therapies at the National Institutes of Health Clinical Center from before onset of critical illness to recovery. Pearson analysis of daily gene expression signatures revealed marked gene expression changes in peripheral blood leukocytes that correlated with changes in serum and peripheral blood leukocytes, viral load, antibody responses, coagulopathy, multiple organ dysfunction, and then recovery. This study revealed marked shifts in immune and antiviral responses that preceded changes in medical condition, indicating that clearance of replicating Ebola virus from peripheral blood leukocytes is likely important for systemic viral clearance.

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A detailed understanding of serological immune responses to Ebola and Marburg virus infections will facilitate the development of effective diagnostic methods, therapeutics, and vaccines. We examined antibodies from Ebola or Marburg survivors 1 to 14 years after recovery from disease, by using a microarray that displayed recombinant nucleoprotein (NP), viral protein 40 (VP40), envelope glycoprotein (GP), and inactivated whole virions from six species of filoviruses. All three outbreak cohorts exhibited significant antibody responses to antigens from the original infecting species and a pattern of additional filoviruses that varied by outbreak. NP was the most cross-reactive antigen, while GP was the most specific. Antibodies from survivors of infections by Marburg marburgvirus (MARV) species were least cross-reactive, while those from survivors of infections by Sudan virus (SUDV) species exhibited the highest cross-reactivity. Based on results revealed by the protein microarray, persistent levels of antibodies to GP, NP, and VP40 were maintained for up to 14 years after infection, and survival of infection caused by one species imparted cross-reactive antibody responses to other filoviruses.

RESUMO

Robust humoral and cellular immunity are critical for survival in humans during an ebolavirus infection. However, the interplay between these two arms of immunity is poorly understood. To address this, we examined residual immune responses in survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). Cytokine and chemokine expression levels in SUDV stimulated whole blood cultures were assessed by multiplex ELISA and flow cytometry. Antibody and corresponding neutralization titers were also determined. Flow cytometry and multiplex ELISA results demonstrated significantly higher levels of cytokine and chemokine responses in survivors with serological neutralizing activity. This correspondence was not detected in survivors with serum reactivity to SUDV but without neutralization activity. This previously undefined relationship between memory CD4 T cell responses and serological neutralizing capacity in SUDV survivors is key for understanding long lasting immunity in survivors of filovirus infections.

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Induction of lymphopenia has been exploited therapeutically to improve immune responses to cancer therapies and vaccinations. Whereas IL-15 has well-established roles in stimulating lymphocyte responses after lymphodepletion, the mechanisms regulating these IL-15 responses are unclear. We report that cell surface IL-15 expression is upregulated during lymphopenia induced by total body irradiation (TBI), cyclophosphamide, or Thy1 Ab-mediated T cell depletion, as well as in RAG(-/-) mice; interestingly, the cellular profile of surface IL-15 expression is distinct in each model. In contrast, soluble IL-15 (sIL-15) complexes are upregulated only after TBI or αThy1 Ab. Analysis of cell-specific IL-15Rα conditional knockout mice revealed that macrophages and dendritic cells are important sources of sIL-15 complexes after TBI but provide minimal contribution in response to Thy1 Ab treatment. Unlike with TBI, induction of sIL-15 complexes by αThy1 Ab is sustained and only partially dependent on type I IFNs. The stimulator of IFN genes pathway was discovered to be a potent inducer of sIL-15 complexes and was required for optimal production of sIL-15 complexes in response to Ab-mediated T cell depletion and TBI, suggesting products of cell death drive production of sIL-15 complexes after lymphodepletion. Lastly, we provide evidence that IL-15 induced by inflammatory signals in response to lymphodepletion drives lymphocyte responses, as memory CD8 T cells proliferated in an IL-15-dependent manner. Overall, these studies demonstrate that the form in which IL-15 is expressed, its kinetics and cellular sources, and the inflammatory signals involved are differentially dictated by the manner in which lymphopenia is induced.

RESUMO

BACKGROUND: Human monkeypox is a zoonotic disease endemic to parts of Africa. Similar to other orthopoxviruses, virus and host have considerable interactions through immunomodulation. These interactions likely drive the establishment of a productive infection and disease progression, resulting in the range of disease presentations and case fatality rates observed for members of the Orthopoxvirus genus. OBJECTIVES: Much of our understanding about the immune response to orthopoxvirus infection comes from either in vitro or in vivo studies performed in small animals or non-human primates. Here, we conducted a detailed assessment of cytokine responses to monkeypox virus using serum from acutely ill humans collected during monkeypox active disease surveillance (2005-2007) in the Democratic Republic of the Congo. STUDY DESIGN: Nineteen serum samples that were from patients with confirmed monkeypox virus infections were selected for cytokine profiling. Cytokine profiling was performed on the Bio-Rad Bioplex 100 system using a 30-plex human cytokine panel. RESULTS: Cytokine profiling revealed elevated cytokine concentrations in all samples. Overproduction of certain cytokines (interleukin [IL]-2R, IL-10, and granulocyte macrophage-colony stimulating factor were observed in patients with serious disease (defined as >250 lesions based on the World Health Organization scoring system). CONCLUSIONS: The data suggest that cytokine modulation affects monkeypox disease severity in humans.

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Recovery from ebolavirus infection in humans is associated with the development of both cell-mediated and humoral immune responses. According to recent studies, individuals that did not survive infection with ebolaviruses appear to have lacked a robust adaptive immune response and the expression of several early innate response markers. However, a comprehensive protective immune profile has yet to be described. Here, we examine cellular memory immune responses among survivors of two separate Ebolavirus outbreaks (EVDs) due to Sudan virus (SUDV) infection in Uganda-Gulu 2000-2001 and Kibaale 2012. Freshly collected blood samples were stimulated with inactivated SUDV, as well as with recombinant SUDV or Ebola virus (EBOV) GP (GP1-649). In addition, ELISA and plaque reduction neutralization assays were performed to determine anti-SUDV IgG titers and neutralization capacity. Cytokine expression was measured in whole blood cultures in response to SUDV and SUDV GP stimulation in both survivor pools, demonstrating recall responses that indicate immune memory. Cytokine responses between groups were similar but had distinct differences. Neutralizing, SUDV-specific IgG activity against irradiated SUDV and SUDV recombinant proteins were detected in both survivor cohorts. Furthermore, humoral and cell-mediated crossreactivity to EBOV and EBOV recombinant GP1-649 was observed in both cohorts. In conclusion, immune responses in both groups of survivors demonstrate persistent recognition of relevant antigens, albeit larger cohorts are required in order to reach greater statistical significance. The differing cytokine responses between Gulu and Kibaale outbreak survivors suggests that each outbreak may not yield identical memory responses and promotes the merits of studying the immune responses among outbreaks of the same virus. Finally, our demonstration of cross-reactive immune recognition suggests that there is potential for developing cross-protective vaccines for ebolaviruses.

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Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8âº and CD8â»). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineageâ»Sca-1âºc-Kitâº) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15âºCD8âº and IL-15(â»/low)CD8â» DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.

RESUMO

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

RESUMO

Invariant NKT (iNKT) cells are an innate type of T cells, which respond rapidly on activation. iNKT cells acquire these innate-like abilities during development; however, the signals driving development and functional maturation remain only partially understood. Because interleukin-15 (IL-15) is crucial for iNKT development and is delivered by transpresentation, we set out to identify the cell types providing IL-15 to developing iNKT cells and determine their role at the various states of development and maturation. We report here that transpresentation of IL-15 by parenchymal cells was crucial for generating normal number of iNKTs in the thymus, whereas both hematopoietic and parenchymal cells regulated iNKT cell numbers in the periphery, particularly in the liver. Specifically, dendritic cells contributed to peripheral iNKT cell numbers by up-regulating Bcl-2 expression and promoting extrathymic iNKT cell ex-pansion and their homeostatic proliferation. Whether IL-15 affects functional maturation of iNKT cells was also examined. In IL-15Rα(-/-) mice, CD44(High)NK1.1(+) iNKT cells displayed decreased T-bet expression and in response to α-galactosylceramide, had deficient interferon-Î³ expression. Such defects could be reversed by exogenous IL-15 signals. Overall, these studies identify stage-specific functions of IL-15, which are determined by the tissue microenvironment and elucidate the importance of IL-15 in functional maturation.

RESUMO

This study tested the hypothesis that individual myeloid subsets have a differential ability to maintain memory CD8 T cells via IL-15. Although DCs support IL-15-mediated homeostasis of memory CD8 T cells in vivo, whether various DC subsets and other myeloid cells similarly mediate homeostasis is unknown. Therefore, we studied the ability of different myeloid cells to maintain memory CD8 T cells in vitro. Using an in vitro cocoulture system that recapitulated known roles of DCs and IL-15 on memory CD8 T cells, all in vitro-derived or ex vivo-isolated DCs maintained CD8 T cells better than rIL-15 alone, and FLT-3L-DCs are the most efficient compared with GM-DCs, BM-derived macrophages, or freshly isolated DCs. Although FLT-3L-DCs were the least effective at inducing CD8 T cell proliferation, FLT-3L-DCs promoted better CD8 T cell survival and increased Bcl-2 and MCL-2 expression in CD8 T cells. T cell maintenance correlated only partially with DC expression of IL-15Ralpha and IL-15, suggesting that DCs provided additional support signals. Indeed, in the absence of IL-15 signals, CD70/CD27 further supported CD8 T cell maintenance. IFN-alpha enhanced CD70 expression by DCs, resulting in increased proliferation of CD8 T cells. Overall, this study supports our hypothesis by demonstrating that specific DC subtypes had a greater capacity to support memory CD8 T cell maintenance and did so through different mechanisms. Furthermore, this study shows that IL-15 trans-presentation can work in conjunction with other signals, such as CD70/CD27 interactions, to mediate CD8 T cell homeostasis efficiently.

RESUMO

Interleukin (IL)-15 is a cytokine that acts on a wide range of cell types but is most crucial for the development, homeostasis, and function of a specific group of immune cells that includes CD8 T cells, NK cells, NKT cells, and CD8 alpha alpha intraepithelial lymphocytes. IL-15 signals are transmitted through the IL-2/15R beta and common gamma (gamma C) chains; however, it is the delivery of IL-15 to these signaling components that is quite unique. As opposed to other cytokines that are secreted, IL-15 primarily exists bound to the high affinity IL-15R alpha. When IL-15/IL-15R alpha complexes are shuttled to the cell surface, they can stimulate opposing cells through the beta/gamma C receptor complex. This novel mechanism of IL-15 delivery has been called trans-presentation. This review discusses how the theory of trans-presentation came to be, evidence that it is the major mechanism of action, the current understanding of the cell types thought to mediate trans-presentation, and possible alternatives for IL-15 delivery.

RESUMO

IL-15 is a key component that regulates the development and homeostasis of NK cells and is delivered through a mechanism termed trans-presentation. During development, multiple events must proceed to generate a functional mature population of NK cells that are vital for tumor and viral immunity. Nevertheless, how IL-15 regulates these various events and more importantly what cells provide IL-15 to NK cells to drive these events is unclear. It is known dendritic cells (DC) can activate NK cells via IL-15 trans-presentation; however, the ability of DC to use IL-15 trans-presentation to promote the development and homeostatic maintenance of NK cell has not been established. In this current study, we show that IL-15 trans-presentation solely by CD11c(+) cells assists the in vivo development and maintenance of NK cells. More specifically, DC-mediated IL-15 trans-presentation drove the differentiation of NK cells, which included the up-regulation of the activating and inhibitory Ly49 receptors. Although these cells did not harbor a mature CD11b(high) phenotype, they were capable of degranulating and producing IFN-gamma upon stimulation similar to wild-type NK cells. In addition, DC facilitated the survival of mature NK cells via IL-15 trans-presentation in the periphery. Thus, an additional role for NK-DC interactions has been identified whereby DC support the developmental and homeostatic niche of NK cells.

RESUMO

Type I IFNs, including IFN-alpha, enhance Ag presentation and promote the expansion, survival, and effector function of CD8(+) CTL during viral infection. Because these are ideal characteristics for a vaccine adjuvant, we examined the efficacy and mechanism of exogenous IFN-alpha as an adjuvant for antimelanoma peptide vaccination. We studied the expansion of pmel-1 transgenic CD8(+) T cells specific for the gp100 melanocyte differentiation Ag after vaccination of mice with gp100(25-33) peptide in IFA. IFN-alpha synergized with peptide vaccination in a dose-dependent manner by boosting relative and absolute numbers of gp100-specific T cells that suppressed B16 melanoma growth. IFN-alpha dramatically increased the accumulation of gp100-specific, IFN-gamma-secreting, CD8(+) T cells in the tumor through reduced apoptosis and enhanced proliferation of Ag-specific CD8(+) T cells. IFN-alpha treatment also greatly increased the long-term maintenance of pmel-1 CD8(+) T cells with an effector memory phenotype, a process that required expression of IFN-alpha receptor on the T cells and IL-15 in the host. These results demonstrate the efficacy of IFN-alpha as an adjuvant for peptide vaccination, give insight into its mechanism of action, and provide a rationale for clinical trials in which vaccination is combined with standard-of-care IFN-alpha therapy for melanoma.

RESUMO

Interleukin-15 (IL-15) is crucial for the development of naive and memory CD8 T cells and is delivered through a mechanism called transpresentation. Previous studies showed that memory CD8 T cells require IL-15 transpresentation by an as yet unknown cell of hematopoietic origin. We hypothesized that dendritic cells (DCs) transpresent IL-15 to CD8 T cells, and we examined this by developing a transgenic model that limits IL-15 transpresentation to DCs. In this study, IL-15 transpresentation by DCs had little effect on restoring naive CD8 T cells but contributed to the development of memory-phenotype CD8 T cells. The generation of virus-specific, memory CD8 T cells was partially supported by IL-15Ralpha(+) DCs through the preferential enhancement of a subset of KLRG-1(+)CD27(-) CD8 T cells. In contrast, these DCs were largely sufficient in driving normal homeostatic proliferation of established memory CD8 T cells, suggesting that memory CD8 T cells grow more dependent on IL-15 transpresentation by DCs. Overall, our study clearly supports a role for DCs in memory CD8 T-cell homeostasis but also provides evidence that other hematopoietic cells are involved in this function. The identification of DCs fulfilling this role will enable future studies to better focus on mechanisms regulating T-cell homeostasis.

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